The last post was about Conjugation. In this post (a continuation to Conjugation) we discuss about the Interrupted Mating technique and Mapping of genes in bacterial chromosome using the same.
As seen in the last post, Hfr is formed when the F plasmid is integrated into the bacterial chromosome (fig 1).
In the integrated state, too, the genes of F factor are functional and can bring about the formation of the F pilus, replication and transfer of the plasmid in the integrated form (fig 2.1& 2.2). However, the transfer of the entire integrated F factor requires a long time and hence happens at a very low frequency (⁓1 in 10,000) due to random breakage (fig 2.3). Therefore, in most of the cases, only the initial part of F factor (adjoining oriT) and few bacterial genes and not the entire integrated F factor are transferred from the Hfr strain into the F- cells (fig 2.4). The donor chromosomal genes transferred, get incorporated into the genome of the F- cell, resulting into the recombinant cell (fig 2.5). Hence the recipient, a F- cell is mostly not converted to F+ cell but a F- recombinant cells.
• Linkage Determination using Conjugation.
More information on Conjugation were obtained by experiments conducted by Elie Wollman and Francois Jacob. In one of their experiments, they mixed two strains, the Hfr H (H: isolated by Hayes) and a F- recipient cell, in liquid media favourable for growth of both the bacteria. The bacteria were of following genotypes:
Hfr : str s, azi r, ton r, lac+, gal+
F- : str r, azi s, ton s, lac -, gal –
(str s: sensitive to streptomycin, azi r: resistant to , lac+: could utilize lactose as sole source, gal+: could utilize galactose when solo source of carbon.)
The conditions favourable for the conjugation were provided. After a few minutes, the bacterial cells in the liquid media were agitated in a kitchen blender to separate the paired cells or in other words to interrupt the conjugative mating. This procedure is called interrupted mating (fig 3). These steps were repeated at regular intervals from the initiation of conjugation.
The cells were then transferred onto the selective medium containing streptomycin to remove the streptomycin sensitive Hfr donor cells. The cells growing on the media containing the streptomycin resistant (str r) cells were then investigated for the other genes. The str r recipients cells having a gene from the donor, are the ones in which the conjugation had initiated. These cells are called exconjugants.
In this experiment, they observed that the recombinants gained azi r gene after around 8 minutes, ton r after 10 minutes, lac+ after 18 minutes and gal + after 25 minutes. Also, the percentages of exconjugant colonies were 90% for azi r, 80% for ton r, 40% for lac+, and 20% for gal+ (fig 4).
Hence, each gene entered the F- cell at a particular time and the percentage of the cells receiving the early entering genes were much higher than the cells with the later entering genes.
These observations led to the conclusions that:
- The transfer of the Hfr initiated at a particular point, hence each gene was transferred at a particular time after the initiation of the conjugation. This point of initiation is termed as origin of transfer.
- The transfer of the genes took place in a linear manner; hence the later genes were transferred to a smaller number of bacterial cells compared to the initial genes as the mating pairs were separated during the transfer process.
These experiments made it clear that such experiments could be used to predict the order of the genes in the bacterial chromosome (early to late entering genes). Hence, a bacterial chromosome map can be prepared and the distance between the genes can be measured in terms of the time in minutes of the chromosome transfer. Wollman and Jacob constructed linkage maps from the interrupted-mating results, at the time, without knowledge of the physical basis. Eg. In the fig 5, Bacterial map with the order of the genes entering the F- recipient cells with respect to time are mapped (using data from fig 4).
• Chromosome circularity and integration of F factor
Interrupted mating technique also gave early evidences of the bacterial genome being circular was obtained, which has been confirmed by various other studies later.
It was observed that when interrupted-mating studies were performed on different Hfr strains, the sequence of the genes entering the F- cells varied considerably from strain to strain. The time at which a particular gene entered was not fixed, however it always entered between the same adjoining genes.
For eg. in the fig 7b, the sequence of genes vary but the gene ‘pur’ is always between ‘lac’ and ‘gal’.
Allan Campbell concluded that this was because the chromosome of F+ donor was circular.
As the Hfr chromosomes is generated when the circular F plasmid gets integrated into the circular bacterial chromosome, the difference in the sequences of the genes in the different Hfr strains arise due to the difference in the location and the orientation of the F factor insertion (Fig 7a).
As the transfer always begins from the origin of transfer, and the location of the F plasmid insertion determines where transfer begins. Hence the adjacent genes vary and so does the sequence of genes transfer.
The orientation of the plasmid within the bacterial chromosome determines whether transfer will occur clockwise or counterclockwise (Fig 7b).
Hence the conjugative transfer in Hfr cells can be subjected to interrupted mating technique and used to map the entire circular bacterial chromosome.
(Just for info: here’s an example of experiment carried out in 1973 to map a mutation in E.coli K12 using interrupted mating technique).
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Have a nice day!
Also read other posts by The Biotech Notes:
Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000. Bacterial conjugation. https://www.ncbi.nlm.nih.gov/books/NBK21942/